EP2843411A1 - Luciferase-Signalverbesserungszusammensetzungen - Google Patents

Luciferase-Signalverbesserungszusammensetzungen Download PDF

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Publication number
EP2843411A1
EP2843411A1 EP14187757.1A EP14187757A EP2843411A1 EP 2843411 A1 EP2843411 A1 EP 2843411A1 EP 14187757 A EP14187757 A EP 14187757A EP 2843411 A1 EP2843411 A1 EP 2843411A1
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Prior art keywords
luciferase
secreted
reagent composition
activity
buffer
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French (fr)
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EP2843411B1 (de
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Marco Peter Leu
John Daly
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Gene Stream Pty Ltd
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Gene Stream Pty Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Definitions

  • the present invention relates generally to reagents and compositions for use in reactions catalysed by luciferase enzymes, for example in luciferase-based gene reporter assays and applications in which luciferase enzymes are utilised as a detectable and/or quantifiable label bound to a molecule such as antibodies for immunocytochemical assays or enzyme-linked immunosorbent assays (ELISA).
  • the invention also provides methods and compositions for, inter alia, increasing the sensitivity and/or improving the kinetics of luciferase-catalysed reactions.
  • Reporter gene assays represent an important tool in studies of gene expression, permitting an understanding of what controls the expression of a gene of interest e.g., DNA sequences, transcription factors, RNA sequences, RNA-binding proteins, signal transduction pathways and specific stimuli.
  • reporter assays can be used to identify nucleic acid regions important in gene regulation. Such regions and/or the factors that bind or modulate them may serve as potential targets for therapeutic intervention in the treatment or prevention of human diseases. Reporter assays can also be used to screen drugs for their ability to modify gene expression.
  • reporter assays are used to identify a gene promoter region or specific elements within a promoter, such as transcription factor binding sites or other regulatory elements. Alternatively, such assays are used to study the response of a promoter or regulatory element to various stimuli or agents. In some applications, the reporter constructs used in the assay, or transacted cells, are introduced into an organism to study promoter function in vivo. Further, reporter assays can be used to study or measure signal transduction pathways upstream of a specific promoter.
  • nucleic acids to be interrogated are cloned into reporter plasmids in a location so as to permit the regulation of transcription of a downstream reporter gene, and thus expression of a reporter protein encoded by the reporter gene.
  • the reporter protein should be distinguishable from endogenous proteins present in the cell in which the reporter plasmid is transacted for ease of detection, and preferably expression of the reporter protein should be readily quantifiable.
  • the reporter protein is quantified in an appropriate assay and often expressed relative to the level of a control reporter driven by a ubiquitous promoter such as, for example, the promoter SV40.
  • control reporter must be distinguishable from the test reporter and is generally contained on a separate vector that is co-transfected with the test vector and used to control for transfection efficiency.
  • assays are based on the premise that cells take up proportionally equal amounts of both vectors.
  • a variety of different applications for gene reporter assays involve measuring a change in gene expression over time or after addition of a compound, such as a drug, ligand, hormone etc. This is of particular importance in drug screening. Following the addition of the drug, detecting a measurable change in levels of the reporter protein may be delayed and diluted as changes in expression levels are transmitted through mRNA to protein.
  • a significant advance in such applications recently made by the present applicant is the combined use of mRNA-and protein-destabilizing elements in the reporter vector to improve the speed and magnitude of response, as described in co-pending US Patent Application No. 10/658,093 , the disclosure of which is incorporated herein by reference.
  • reporter gene assay systems are commercially available utilising different detectable reporter proteins, the most common being chloramphenicol transferase (CAT), ⁇ galactosidase ( ⁇ -gal), secreted alkaline phosphatase, and various fluorescent proteins and luciferases.
  • CAT chloramphenicol transferase
  • ⁇ -gal ⁇ galactosidase
  • secreted alkaline phosphatase ase
  • fluorescent proteins and luciferases various fluorescent proteins and luciferases.
  • Luciferase is the most commonly used reporter protein for in vitro assay systems. Luciferases are enzymes capable of bioluminescence and are found naturally in a range of organisms. In commercially available assay systems, luciferases can be divided into those which utilise D-luciferin as a substrate and those which utilise coelenterazine as a substrate. The most widely employed example of the former is firefly luciferase, an intracellular enzyme. Additional examples of luciferases utilising D-luciferin include other members of Coleoptera, such as click beetles and railroad worms. Luciferases may also be distinguished on the basis of whether the organism from which they are derived is terrestrial or aquatic (typically marine).
  • Luciferases utilising coelenterazine as a substrate are typically derived from marine animals such as the soft coral Renilla or the copepod Gaussia, whereas D-luciferin-utilising luciferases are typically derived from terrestrial animals.
  • a further means of distinguishing luciferases is on the basis of whether they are secreted or non-secreted in their native state; i.e. in the organism from which they are derived.
  • Luciferases derived from terrestrial organisms are typically non-secreted (intracellular), whilst those derived from marine organisms may be secreted or non-secreted (intracellular).
  • Renilla luciferase is intracellular, whereas Gaussia luciferase in its native state is a secreted enzyme.
  • the secretion of luciferases by marine organisms is thought to be a protective response designed to distract approaching predators.
  • Other secreted luciferases include those from Metridia longa , Vargula hilgendorfii, Oplophorus gracilirostris, Pleuromamma xiphias, Cypridina noctiluca and other members of Metridinidae.
  • Vargula luciferase utilises a substrate that is different to coelenterazine or D-luciferin.
  • Another class of luciferase is those derived from dinoflagellates.
  • Luciferase-based assay systems may employ more than one luciferase, typically of different origin and each utilising a different substrate, enabling both test and control reporter to be measured in the same assay.
  • a putative promoter element is cloned upstream of a firefly luciferase reporter gene such that it drives expression of the luciferase gene.
  • This plasmid is transiently transfected into a cell line, along with a control plasmid containing the Renilla luciferase gene driven by the SV40 promoter.
  • First luciferin is added to activate the firefly luciferase, activity of this reporter is measured, and then a "quench and activate" reagent is added.
  • This "quench and activate" reagent contains a compound that quenches the luciferin signal and also contains coelenterazine to activate the Renilla luciferase, the activity of which is then measured.
  • the level of firefly luciferase activity is dependent not only on promoter activity but also on transfection efficiency. This varies greatly, depending on the amount of DNA, the quality of the DNA preparation and the condition of the cells.
  • the co-transfected control plasmid ( Renilla luciferase driven by a suitable promoter such as the SV40 promoter) is used to correct for these variables, based on the premise that Renilla luciferase activity is proportional to the amount of firefly luciferase-encoding plasmid taken up by the cells.
  • the Renilla luciferase may be used to control for other variables, such as cell number, cell viability and/or general transcriptional activity; or may be used to determine whether a particular treatment or compound applied to the cells affects both promoters or is specific to one of them.
  • Luciferase-based assay systems in particular those utilising one or more intracellular luciferases, often employ two buffers, a lysis buffer and an assay buffer.
  • the lysis buffer is added to the cells first to lyse the cells and thus release luciferase, facilitating subsequent measurement.
  • An assay buffer containing the luciferase substrate and any cofactors is then added, after which measurement of luciferase activity is taken. Measurement may be made immediately (i.e. within seconds) of the addition of the assay buffer (so-called “flash” reaction), or minutes or hours later (so-called “glow” reactions) by using "glow” reagents in the assay buffer that keep the light signal stable for an extended period of time.
  • Flash reactions provide the highest signal strength (light units per second) and thereby have the advantage of providing the highest sensitivity. Glow reactions are particularly advantageous in applications where, for example, the user does not have a suitable luminometer (equipped with injectors) readily available or in some high throughput screening applications where batch-processing requires a delay between injection and measurement.
  • Secreted luciferases are measured in samples of the conditioned medium surrounding the test cells. As such, lysis buffers are not used with secreted luciferases.
  • reagents, reaction compositions and kits that provide improved sensitivity in luciferase reactions; that is, a signal strength of greater intensity than is achievable with existing reagents.
  • the reporter gene assayed provides only low levels of luciferase in the cells of interest, for example, where the promoter being studied has only low activity, and/or where the cells of interest are difficult to transfect/transduce with the reporter vector.
  • Increased sensitivity would also facilitate the miniaturization of reporter assays by reducing the minimum number of cells required to yield a signal strength that can be reliably measured.
  • the present invention provides reagent compositions, and kits comprising such compositions, for use in determining the amount or activity of a luciferase enzyme in a sample.
  • the present invention further provides methods for using the reagent compositions and for determining the amount or activity of a luciferase enzyme in a sample.
  • the present invention is predicated, in part, on the inventors' surprising findings that various modifications of reagent compositions can have profound impact on the activity of certain classes of luciferase enzymes and on the development of novel reagent compositions which permit, inter alia, the generation of higher sensitivity (stronger flash phase), a reduced rate of luminescent signal decay (a more stable glow phase), reduced lysis time and/or assay time, and improved stability of enzymatic activity or potential over time than is achievable using currently available compositions.
  • luciferases that are secreted in their native form have properties that are distinct from the more typically used intracellular luciferases and as such have quite distinct requirements with respect to reagent compositions.
  • these normally secreted luciferases are expressed intracellularly, the inventors discovered that they become unsuitable for use with any currently available reagent compositions.
  • the inventors subsequently identified the key features and components required to make reagents that are suitable for use with such luciferases when used alone or in combination with a different class of luciferase.
  • the present invention provides a reagent composition for use in determining the amount and/or activity of luciferase in a sample, wherein the reagent composition permits generation of an enhanced luminescent signal, a reduced rate of luminescent signal decay from the luciferase and/or improved stability of luciferase activity over time in cell lysates.
  • the reagent composition provides an environment suitable to facilitate conversion of the luciferase into an active conformation.
  • the luciferase may be secreted or non-secreted and may be derived from a luciferase that is secreted or non-secreted in its native form.
  • the luciferase is derived from a luciferase that is secreted in its native form.
  • the luciferase is a non-secreted luciferase that is a modified form of a luciferase which is secreted in its native form.
  • the non-secreted luciferase may be expressed in the cytoplasm or other cellular compartment, typically wherein the cellular compartment provides a reducing environment.
  • the luciferase may utilise any known luciferase substrate, such as, for example, luciferin or coelenterazine.
  • the luciferase utilises coelenterazine as substrate and is of marine origin.
  • the luciferase of marine origin may be derived, for example, from Gaussia spp., Pleuromamma spp., Metridia spp., Cypridina spp. or Oplophorus spp.
  • the luciferase may be a variant or derivative of a naturally occurring luciferase.
  • the reagent composition may comprise one or more chelators, bromide anions, a non- ionic detergent at a concentration of less than 1% or a zwitterionic detergent, at least one oxidising agent or combination of oxidising and reducing agents, and/or have a pH of above about 8.
  • the chelator is a divalent metal chelator.
  • the divalent metal chelator may, for example, be selected from EDTA, CDTA and EGTA.
  • the divalent metal chelator is EDTA1 present at a concentration of at least 0.1mM, more preferably between about 1 mM and 30 mM, more typically between about 4 mM and about 15 mM.
  • the detergent may be a zwitterionic or more preferably a non-ionic, detergent.
  • the detergent is preferably at a final concentration of less than about 1%.
  • the detergent may be present at a concentration of between about 0.05% and about 0.2% when first contacted with the sample or cell,
  • the detergent may be selected from, for example, Triton X-100, NP101 or NP40. In a particular embodiment the detergent is NP40.
  • the oxidising agent or combination of oxidising and reducing agents result in oxidation of the luciferase thereby facilitating the adoption of an active conformation by the luciferase.
  • the reducing agent may comprise a thiol group.
  • the composition comprises a redox buffer combination such as a mixture of oxidised and reduced glutathione.
  • the reagent composition has a pH of above about 8, more typically between about 8 and about 9, or more typically between about 8.4 and 8.8.
  • the bromide anions may be provided in the form of one or more bromide salts.
  • the bromide salts may be, for example, sodium bromide, potassium bromide or rubidium bromide.
  • the bromide anion is present at a concentration of at least about 1 mM, generally between about 1 mM and about 500 mM.
  • the present invention provides a reagent composition for determining the amount and/or activity of a recombinant luciferase in a sample, the reagent composition comprising one or more chelators, wherein the recombinant luciferase is a non-secreted variant of a luciferase that is secreted in its native form.
  • the chelator may be a divalent metal chelator.
  • the chelator may, for example, be selected from EDTA, CDTA and EGTA.
  • the divalent metal chelator is EDTA, present at a concentration of at least 0.ImM1 more preferably between about 1 mM and 30 mM, more typically between about 4 mM and about 15 mM.
  • the reagent composition may further comprise bromide anions, a non-ionic detergent at a concentration of less than 1% or a zwitterionic detergent, at least one oxidising agent or combination of oxidising and reducing agents, and/or have a pH of above about 8.
  • the reagent composition may further comprise the luciferase substrate.
  • the present invention provides a reagent composition for determining the amount and/or activity of a recombinant luciferase in a sample, the reagent composition comprising bromide anions, wherein the recombinant luciferase is a non-secreted variant of a luciferase that is secreted in its native form.
  • the bromide anions may be provided in the form of one or more bromide salts.
  • the bromide salts may be selected from sodium bromide, potassium bromide or rubidium bromide.
  • the bromide anion is present at a concentration of at least about 1 mM, generally between about 1 mM and about 500 mM.
  • the reagent composition may further comprise one or more chelators, a non-ionic detergent at a concentration of less than 1 % or a zwitterionic detergent, at least one oxidising agent or combination of oxidising and reducing agents, and/or have a pH of above about 8.
  • the reagent composition may further comprise the luciferase substrate.
  • the present invention provides a method for determining the amount and/or activity of luciferase in a cell or sample of cells, the method comprising:
  • the composition for converting the luciferase from an inactive state or conformation to an active state or conformation provides a chelator and/or a redox environment suitable for conversion of the luciferase into an active conformation.
  • the redox environment may be suitable for or enable oxidation of the luciferase.
  • the reagent composition may comprise one or more of bromide anions, a non-ionic detergent at a concentration of less than 1 % or a zwitterionic detergent, at least one oxidising agent or combination of oxidising and reducing agents, and have a pH of above about 8.
  • the reagent composition may be a composition of any one of the first to the third aspects.
  • the reagent composition may further comprise the luciferase substrate.
  • the luciferase may be a recombinant luciferase that is a non-secreted variant of a luciferase that is secreted in its native form.
  • the present invention provides a method for determining the amount and/or activity of a recombinant luciferase in a cell or sample of cells, the method comprising:
  • the reagent composition may comprise a detergent, such that steps (a) and (b) may be combined in a single step.
  • the reagent composition comprises the luciferase substrate such that steps (b) and (c) may be combined in a single step.
  • the reagent composition may be a composition of any one of the first to the third aspects.
  • present invention provides a method for increasing the bioluminescent signal generated by a luciferase enzyme, the method comprising contacting the luciferase with an effective amount of a reagent composition that provides an environment that enables or promotes conversion of the luciferase into an active state or conformation such as a suitable redox environment.
  • the reagent composition comprises one or more of a bromide salt, a non- ionic detergent at a concentration of less than 1%, a divalent metal chelator, preferably at a concentration of at least 1mM, at least one oxidising agent or combination of oxidising and reducing agents, and a pH of above about 8.
  • the present invention provides a method for increasing the bioluminescent signal generated by a luciferase enzyme, the method comprising contacting the luciferase with an effective amount of one or more divalent metal chelators.
  • the divalent metal chelator may, for example, be selected from EDTA, CDTA and EGTA.
  • the divalent metal chelator is EDTA, present at a concentration of at least 0.ImM, more preferably between about 1 mM and 30 mM, more typically between about 4 mM and about 15 mM.
  • the present invention provides a method for increasing the bioluminescent signal generated by a luciferase enzyme, the method comprising contacting the luciferase with an effective amount of bromide anions, typically in the form of a bromide salt.
  • the present invention provides a method of reducing the rate of decay of the bioluminescent signal generated by a luciferase enzyme, the method comprising contacting the luciferase with an effective amount of a reagent composition wherein the reagent composition provides an environment suitable to facilitate conversion of the luciferase into an active state or conformation, such as a suitable redox environment.
  • bioluminescent signal is prolonged during a phase beginning several minutes after addition of substrate.
  • the present invention provides a method of reducing the rate of decay of the bioluminescent signal generated by a luciferase enzyme, the method comprising contacting the luciferase with one or more divalent metal chelators.
  • the divalent metal chelator may, for example, be selected from EDTA, CDTA and EGTA.
  • the divalent metal chelator is EDTA, present at a concentration of at least 0.1 mM, more preferably between about 1 mM and 30 mM, more typically between about 4 mM and about 15 mM.
  • the present invention provides a method of reducing the rate of decay of the bioluminescent signal generated by a luciferase enzyme, the method comprising contacting the luciferase with an effective amount of bromide, typically in the form of a bromide salt.
  • the luciferase is a recombinant luciferase that is a non-secreted variant of a luciferase that is secreted in its native form.
  • the reduce time required to achieve optimal or stable luciferase activity may result from, or be associated with, reduced lysis time and/or assay time, and improved stability of enzymatic activity or potential over time.
  • the present invention provides a kit for use in assaying the amount and/or activity of a luciferase, the kit comprising at least one reagent composition wherein the reagent composition provides an environment suitable to facilitate conversion of the luciferase into an active conformation.
  • the reagent composition may be a composition of any one of the first to the third aspects.
  • the luciferase may be a recombinant luciferase that is a non-secreted variant of a luciferase that is secreted in its native form.
  • the present invention provides a kit for use in assaying the amount and/or activity of a luciferase, the kit comprising bromide ions and a luciferase substrate.
  • the luciferase may be a recombinant luciferase that is a non-secreted variant of a iuciferase that is secreted in its native form.
  • the reagent composition may further comprise an antioxidant.
  • the composition further comprises BSA, protease inhibitors, glycerol, urea or a luciferase substrate.
  • the reagent composition may be in the form of a buffer for lysing cells comprising the luciferase.
  • the lysis buffer may further comprise additional components such as, for example, glycerol and protease inhibitors.
  • the buffering agent may, for example, be Tris, Hepes or a phosphate buffer.
  • the lysis buffer may be in the form of a combined cell lysis/luciferase assay buffer and accordingly, the composition may further comprise a luciferase substrate such as colenterazine.
  • the luciferase is expressed from a reporter gene and the amount or activity of luciferase is determined as part of a reporter gene assay.
  • the reporter gene assay may be part of a multiple luciferase assay.
  • the invention also relates to a reagent composition for use in determining the amount and/or activity of luciferase in a sample, wherein in the presence of the luciferase, the reagent composition provides an environment suitable to facilitate conversion of the luciferase into an active state or conformation.
  • the luciferase may be a recombinant luciferase.
  • the recombinant luciferase may be a non-secreted form of a luciferase that is secreted in its native form.
  • the reagent composition may provide a redox environment suitable to facilitate folding of the luciferase into an active conformation.
  • the reagent composition may provide an environment suitable to facilitate disaggregation of the luciferase and/or separation from interfering proteins and/or unfolding of the luciferase in such a way as to facilitate subsequent refolding of the luciferase into an active state.
  • the environment provided by the reagent composition facilitates a more rapid conversion of inactive luciferase to an active conformation or adoption or maintenance of a more active conformation.
  • the environment may enhance the overall activity of luciferase in a sample by increasing the proportion of luciferase that adopts the most active conformation.
  • the environment may facilitate the maintenance of a state of constant activity of the luciferase in the sample or reduce the time taken for the sample to reach a state of constant activity of the luciferase. Typically, this is achieved by reducing the time taken for the luciferase in the sample to reach its maximum activity.
  • the reagent composition may comprise a zwitterionic or more preferably a non-ionic, detergent.
  • the detergent is preferably at a final concentration of less than about 1%. In one embodiment, the detergent may be present at a concentration of between about 0.05% and about 0.2% when first contacted with the sample or cell.
  • the detergent may be selected from, for example, Triton X-100, NP101 or NP40. In a particular embodiment the detergent is NP40.
  • the reagent composition may comprise at least one suitable oxidising agent or a combination of oxidising and reducing agents.
  • the agent(s) may comprise a thiol group.
  • the agent(s) results in oxidation of the luciferase thereby facilitating the adoption of an active conformation by the luciferase.
  • the composition comprises a redox buffer combination such as a mixture of oxidised and reduced glutathione.
  • the reagent composition may have a pH of above about 8, typically between about 8 and about 9, or more typically between about 8.4 and 8.8.
  • the reagent composition may further comprise one or more chelators such as divalent metal chelators.
  • the chelator may, for example, be selected from EDTA, CDTA and EGTA. In one embodiment, the chelator is EDTA.
  • the chelator is present at a concentration of at least 0.ImM, more preferably between about 1 mM and 30 mM, more typically between about 4 mM and about 15 mM.
  • the reagent composition may further comprise bromide anions, generally in the form of at least one bromide salt.
  • the bromide salt may be, for example, sodium bromide, potassium bromide or rubidium bromide.
  • the bromide anion is present at a concentration of at least about 1 mM, generally between about 1 mM and about 500 mM.
  • the invention also relates to a method for determining the amount or activity of luciferase in a sample, the method comprising:
  • the substrate may be present in the reagent composition of step (a) or in a second composition, optionally also comprising cofactors required for bioluminescent activity of the luciferase, such as CoA, ATP and magnesium.
  • the pH of the second reagent composition may be lower than the pH of the first composition.
  • an element means one element or more than one element.
  • the term “facilitates” means that the reagent composition enables, produces, or promotes such conversion.
  • facilitation of the conversion of luciferase into an active conformation may be passive or active, and direct or indirect.
  • the reagent composition may provide a suitable environment in which conversion of the luciferase into an active conformation may take place.
  • the reagent composition may directly or indirectly promote or otherwise produce or generate such a conversion.
  • An agent, additive or component of a reagent that "facilitates" conversion of a luciferase enzyme is typically one that provides for a more efficient conversion relative to a reagent that lacks the agent but is otherwise identical or substantially equivalent.
  • conversion refers to the folding, disaggregation, re-folding or other modification of a luciferase enzyme in achieving an active state or conformation. Further reference to conversion of a luciferase into an active conformation is to be taken to mean either the conversion from an inactive state or conformation to an active state or conformation, or the conversion from a partially active or less active state or conformation to a more active state or conformation.
  • conversion refers to the structure (for example tertiary or quaternary structure) adopted by the enzyme and which correlates with the ability of the enzyme to catalyse a reaction and generate a bioluminescent signal upon addition of substrate.
  • the actual catalytic activity of the luciferase does not occur in the absence of substrate.
  • a luciferase that has been converted to a more active state or conformation will be capable of generating more luminescence once the substrate is added than would be possible in the absence of such conversion.
  • This increased luminescence would typically be evident in a flash reaction, which is to say that the conversion of a luciferase to an active format is a separate process to the enablement of a prolonged glow by, for example, blocking a negative feedback mechanism that effectively reduces the activity of the luciferase subsequent to its initial catalytic activity in the presence of substrate.
  • enhanced as used herein in the context of the bioluminescent signal intensity of a luciferase means enhanced or increased, qualitatively or quantitatively, signal intensity relative to that achieved in the absence of the reagent composition and/or in the presence of a composition of the prior art.
  • reduced rate of decay is used to indicate a rate of decay of bioluminescent signal in the absence of the reagent composition and/or in the presence of a composition of the prior art.
  • the term "effective amount" includes within its meaning a non-toxic but sufficient amount of a reagent composition to provide the desired effect
  • the exact amount required may vary from case to case depending on factors such as the nature of the sample to be analyzed, the luciferase enzyme used and whether the luciferase is intracellular or secreted, and the constitution of the reagent or composition used. Thus, it is not possible to specify an exact "effective amount”. However, for any given case, an appropriate "effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
  • non-secreted luciferase means a luciferase that is not exported or secreted from a cell into the extracellular environment.
  • non-secreted includes a luciferase retained in the cell in any form, and thus the luciferase may be cytoplasmic or membrane-associated.
  • this secretion and absence of secretion refers to eukaryotic cells.
  • the term "substrate” means the substrate molecule upon which the luciferase acts, excluding any additional cofactors that may be beneficial to, or required for, binding of the luciferase to the substrate and/or catalysis.
  • luciferase catalysed reactions may require or benefit from cofactors such as magnesium, CoA and ATP, however in the context of the present invention such cofactors are not considered to fall within the scope of the term "substrate”.
  • Luciferase "substrates” include for example D-luciferin and coelenterazine.
  • luciferin refers to the substrate D-luciferin and its analogues, which molecules are substrates for luciferases derived from, for example, Coleoptera such as firefly, click beetles and railroad worms.
  • the term luciferin does not encompass coelenterazine, which represents a different luciferase substrate utilized by a distinct class of luciferase (such as those derived from Renilla, Gaussia and Metridia for example).
  • compositions in accordance with embodiments of the invention are particularly, but not exclusively, applicable for use in conjunction with gene reporter assay systems, including those utilising destabilizing elements so as to provide a rapid response in addition to high signal strength.
  • reagent compositions of the invention also find application in other assay systems wherein the amount and/or activity of one or more luciferase enzymes are to be determined.
  • the luciferase may be used as a reporter in an immunoassay or nucleic acid hybridisation assay and thus may be linked to, for example, an antibody or nucleic acid probe.
  • the luciferase may be a reporter or detectable label.
  • the inventors have elucidated unique properties of a particular class of luciferase enzyme; those that are secreted in their native state.
  • the performance of such luciferases in luciferase assays can be substantially improved by using the reagent compositions and methods of the present invention.
  • the inventors have discovered that when such luciferases are modified so as to prevent secretion and provide intracellular expression in target cells, the activity of the modified luciferase is markedly reduced, but can be quickly recovered by using reagent compositions and methods of the invention.
  • the inventors have developed methods and reagent compositions that enable the use of luciferase proteins so modified in a variety of luciferase-catalysed reactions.
  • the inventors have discovered that when a normally secreted luciferase is modified for intracellular expression and expressed in cells such as eukaryotic cells, the activity of the modified luciferase within the live cells is substantially reduced. Following lysis of the cells, the activity remains low but partially recovers over time, albeit very slowly. This poses a number of problems for the use of such modified luciferases in luciferase assays and in effect has prevented their successful commercialisation.
  • the reduced enzymatic activity causes a reduction in bioluminescent signal strength. Longer incubation times (prior to adding substrate to initiate the reaction) provide some improvement to signal strength but also lengthen the time required to complete an assay.
  • luciferase activity is used as a measure of the amount of luciferase protein present in the sample such that it is necessary for each sample to contain the same activity per unit of luciferase protein.
  • luciferase activity is used as a measure of the amount of luciferase protein present in the sample such that it is necessary for each sample to contain the same activity per unit of luciferase protein.
  • the lack of stability of the cell lysates with respect to luciferase activity prevents this, thereby rendering the assay inaccurate.
  • some luciferases show decreased activity over time in cell lysis buffer (e.g. due to protease activity)
  • the modified forms of normally secreted luciferases show substantial increases over time, representing an entirely new hurdle to be overcome if such modified luciferases are to be successfully exploited.
  • the present inventors have for the first time identified components, additives or agents that when contacted with a cell lysate substantially improve the rate and efficiency at which the modified form of a normally secreted luciferase regains its activity or enzymatic potential.
  • examples of such components include chelators, bromide ions, buffers with relatively high pH (e.g. above 8) and oxidising agents and/or a redox buffer. In the presence of such agents the luciferase achieves a high level of activity quickly and thereafter maintains a constant activity. This provides numerous benefits including shorter assay time (reduced time required in lysis buffer), higher sensitivity (greater maximal signal) and improved accuracy (signal less dependent on lysis time on sample-to-sample variations in efficiency of conversion to active form).
  • Reagent compositions of the present invention provide improved kinetics of luciferase-catalysed reactions.
  • very high bioluminescent signal strength in the first few seconds following addition of substrate can be coupled with a prolonged measurable bioluminescent signal (so-called “glow” reaction) for example at least about 10 minutes after initiation. During this "glow” period the signal strength declines only very slowly.
  • Components of reagent compositions of the invention which alone or in combination contribute to the improved kinetics include bromide salts, divalent metal chelators, high pH (at least about 8 or higher) and oxidizing agents, such as a mixture of reduced and oxidized glutathione.
  • Reagent compositions can also include numerous additional components as will be readily appreciated and ascertained by those skilled in the art.
  • various modified cell lysis buffer compositions for example including a bromide salt and/or reduced levels of detergent, provided very high signal strength when used with a modified intracellular Gaussia or Metridia luciferase, compared to the same luciferase used with commercially available Renilla or Gaussia assay reagents.
  • Reagent compositions of the present invention provide high bioluminescent signal strength and prolonged duration (equating to a reduced rate of decay) of bioluminescent signal when compared with presently available compositions.
  • reagent compositions according to embodiments of the invention may provide an environment suitable for converting the luciferase from an inactive state or conformation into an active state or conformation.
  • the change in conformation may comprise a change in protein folding and/or a change in redox state of the luciferase.
  • the conformational change may comprise the formation of one or more disulphide bridges in the luciferase protein.
  • a change in state or conformation of a luciferase enzyme may be desired or required in a number of circumstances.
  • the luciferase may be expressed cytoplasmically as an inactive or partially inactive protein.
  • the luciferase may be a modified non-secreted form of a luciferase that is secreted in its native form.
  • a reagent composition of the invention comprises at least one bromide anion or salt, for example sodium bromide, potassium bromide or rubidium bromide.
  • the concentration of bromide salt may be between about 1 mM and about 500 mM. In one embodiment the concentration is between about 75 mM and about 225 mM.
  • the bromide anion is present at a concentration of at least about 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM or 500 mM.
  • a reagent composition of the invention may comprise a non-ionic detergent at a concentration of less than about 1%.
  • the non-ionic detergent may be present at a concentration of less than about 0.5%, 0.4%, 0.3%, 0.2%, 0.15% or 0.1%.
  • a reagent composition of the invention may comprise a zwitterionic detergent.
  • the suitable concentration of detergent is between about 0.05% and about 0.1%.
  • the non-ionic detergent may be selected from, for example, Triton X-100, NP101 or NP40. In a particular embodiment the detergent is NP40.
  • the zwitterionic detergent may be CHAPS.
  • cells may be lysed by any one or a number of techniques well known in the art. Many of such techniques do not require the use of detergents, but rather cell lysis may be achieved by, for example, sonication, osmotic pressure or other physical pressure. Thus reagent compositions and methods of the invention are not limited to any one particular means of cell lysis.
  • a reagent composition of the invention may have a pH of at least about 8, typically between about 8 and about 10, between about 8 and about 9, or between about 8.4 and about 8.8.
  • the pH may be at least about 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7 or 8.8.
  • a reagent composition of the invention may comprise at least one oxidising agent or a combination of oxidising and reducing agents. Where a combination of oxidising and reducing agents is used, the relative proportions of the agents is typically such that the environment generated in the presence of the luciferase is an oxidising environment when compared with the cytosol of the cell expressing the luciferase.
  • the composition may comprise a 'redox buffer combination' such as a mixture of oxidised and reduced glutathione. Redox pairs that exist and operate within normal eukayotic cells are disclosed, for example, in Table 7 of [ Foyer, CH.
  • Redox buffers may comprise a mixture of an oxidised form of a thiol (such as a disulphide dimer) and a reduced form. The reduced and oxidised thiols may be the same or different.
  • oxidation - reduction (redox) reactions are characterized by a change in oxidation number, usually by a transfer of electrons.
  • the term “oxidation” typically refers to an increase in oxidation state or number (a loss of electrons). Whereas the term “reduction” refers to a decrease in oxidation state or number (a gain of electrons).
  • An "oxidising agent” is sometimes referred to as an electron acceptor, and a “reducing agent” is sometimes referred to as a electron donor.
  • Substances that have the ability to oxidize other substances are said to be “oxidative” and are referred to as “oxidising agents", “oxidants” or “oxidisers”.
  • reducing agents that have the ability to reduce other substances are said to be “reductive” and are referred to as “reducing agents”, “reductants”, or “reducers”.
  • the reductant or reducing agent loses electrons and is oxidised and the oxidant or oxidising agent gains electrons and is reduced.
  • One or more oxidizing agents and/or reducing agents may be present in a reagent composition or in a corresponding reaction mixture in which that reagent is used, in order to provide an overall "oxidising environment” or an overall “reducing environment” in that reagent or reaction mixture.
  • the oxidising agent or combination of oxidising and reducing agents may promote oxidation of the luciferase thereby promoting or otherwise facilitating the adoption of an active (or more active) conformation by the luciferase.
  • the oxidising agent or combination of oxidising and reducing agents may promote oxidation of the luciferase thereby promoting or otherwise facilitating the adoption of an active (or more active) conformation by the luciferase.
  • the oxidising agent may be a sulfhydryl group converting agent that contributes to the generation of an electrochemical potential in the reagent composition such that sulfhydryl groups are oxidised to disulphide bridges whilst the luciferase protein is not denatured.
  • the oxidising agent may be an agent capable of oxidising, directly or indirectly, thiol or cysteine thiol groups in the luciferase protein.
  • reagent compositions of the invention may comprise a combination of oxidising and reducing agents.
  • the oxidising agent may be an agent capable of oxidising, directly or indirectly, thiol or cysteine thiol groups in the luciferase protein
  • the reducing agent may be an agent capable of reducing, directly or indirectly, thiol or cysteine thiol groups in the luciferase protein.
  • thiols as reducing components of redox buffers, are known to affect the rates of thiol-disulphide interchange reactions involved in protein folding.
  • the redox buffer thiols can act as nucleophile, central thiol, or a leaving group.
  • the overall rate of protein folding can be modulated by variation of the thiol component of a redox buffer.
  • Equation 1.0 A general equation describing the oxidation of thiols is shown in equation 1.0: R-SH + R 1 SH -> R-S-S-R 1 + 2H + + 2e - 1.0
  • the oxidising and reducing agents may be different compounds, or alternatively may be the oxidised and reduced forms of the same compound, e.g. the buffer may be comprised of a mixture of: R 5 -SH (reduced form) and R 5 -S-S-R 5 (oxidised form) or may, for example be comprised of: R 6 -SH (reduced form) and R 2 -S-S-R 2 (oxidised form).
  • a suitable thiol for use in a redox buffer system is glutathione which is present as a thiol and a disulphide dimer.
  • the glutathione redox buffer system uses the disulphide GSSG to provide oxidising equivalents and the monothiol GSH to generally catalyse disulphide bond isomerisation.
  • the GSH component of the GSH/GSSG buffer system can be replaced with other thiols.
  • the rate of in vitro folding of disulphide-containing proteins may be increased by utilising a small-molecule aromatic and aliphatic thiols.
  • Monothiols with lower thiol pKa such as N-methylmercaptoacetamide (NMA) or 4-mercaptobenzoic acid form less stable disulphides and can be used at higher concentrations to give faster folding rates than glutathione.
  • NMA N-methylmercaptoacetamide
  • 4-mercaptobenzoic acid form less stable disulphides and can be used at higher concentrations to give faster folding rates than glutathione.
  • the leaving group ability of thiols is inversely correlated to the pKa of the thiol [ Gough, J., D., J. Am. Chem.
  • the thiol concentration for aromatic thiols in redox buffer systems may vary considerably.
  • aromatic thiols such as 2,2'-[(4-mercaptophenyl)imino]bisethanol, in oxidation and thiol disulphide interchange reactions is known [ DeCoIIo1 T. V., J. Org. Chem., 2001, 66, 4244 - 4249 ],
  • Dithiols may also be used as a component of a redox buffer.
  • dithiols can form cyclic disulphides and thus form less stable mixed disulphides.
  • the addition of reduced dithiol ( ⁇ )-trans-1,2-bis(mercaptoacetamido)cyclohexane (BMC or Vectrase-P) to a glutathione redox buffer may, for example, increase the rate and yield of folding of a protein.
  • BMC can catalyse native disulphide bond formation, both in vitro and in vivo.
  • the second thiol of BMC may provide an intramolecular clock for substrate-induced thiol-disulphide exchange.
  • the oxidising agent may be an enzyme such as endoplasmic reticulum oxidoreductin 1 protein (Ero1p). Oxidative protein folding may involve both the oxidation of thiols and the isomerisation of non-native disulphide bonds. Accordingly, isomerase enzymes may also be used as part of a protein oxidising system.
  • a suitable further component of an oxidative buffer is therefore protein disulphide isomerase (PDI) which has a role in catalysing the unscrambling of non-native disulphide bonds in proteins.
  • PDI protein disulphide isomerase
  • Each PDI molecule has two active sites that contain the - Cys-Xaa-Xaa-Cys- sequence.
  • an enzymatic component of a redox buffer may contain the -Cys-Xaa-Xaa-Cys- sequence.
  • Other enzymes that may be used, for example, in a redox buffer system include: thioredoxin, glutaredoxin and peroxiredoxin.
  • the isomerising component may be a mimic or an active fragment of an isomerase enzyme.
  • active dithiol peptide fragments include the Cys-Xaa-Xaa-Cys tetrapeptide and the CysXaaCys tripeptide, wherein Xaa refers to any amino acid residue [ Woycechowsky, K., J., Biochemistry, 2003, 42, 5387 - 5394 ].
  • the tripeptide CysGlyCys (CGC) has been shown to have a disulphide reduction potential close to that of PDI.
  • TCEP tris(2-carboxyethylphosphine)
  • the oxidizing agent may be a mild oxidizing agent, suitable for oxidizing protein thiol groups, particularly cysteine thiol groups of the luciferase.
  • the oxidizing agent may, itself, contain disulphide bridges and may be an amino acid derivative.
  • the reducing agent may itself contain thiol groups and be an amino acid derivative.
  • Suitable oxidising agents and methods include: molecular oxygen, metal ions, Bu 3 SnOMe/FeCl 3 , nitric oxide, halogens (e.g. bromine and iodine), sodium perbortate, borohydride exchange resin (BER)-transition metal salts system, a morpholine iodine complex, PCC, ammonium persulphate, KMnO 4 /CuSO 4 H 2 O 2 , solvent free permanganate, PVP-Na 2 O 4 and cesium fluoride-Celite O 2 system, 2,6-dicarboxypyridinium chlorochromate (2,6-DCPCC) [ Tajbakhsh, M., Tetrahedron Letters, 45, 2004, 1889 - 1893 ]; dimethylsulphoxide [ Shad, S., T., A., Tetrahedron Lett., 2003, 44, 6789 ; Sanz, R, Synthesis, 2002, 856 ; Karimi, B
  • a reagent composition of the invention may comprise one or more chelators.
  • Suitable chelators include but are not limited to divalent metal chelators such as, for example, EDTA, CDTA and EGTA.
  • the chelator may be present at any concentration but preferably at a concentration of between about 0.1 mM and about 50mM, typically at a concentration of between about 0.1 mM and about 3OmM, more typically at a concentration of between about 0.1 mM and about 15mM.
  • the concentration may be at least about 0.1 mM, 0.2mM, 0.5mM, 1mM, 2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM, 10 mM, 12.5 mM or 15 mM.
  • bromide salts including sodium bromide and potassium bromide
  • Gaussia and Metridia luciferases enhance activity of Gaussia and Metridia luciferases (in their native secreted form and as modified, intracellular enzymes) and that activity of both Gaussia luciferases and both Metridia luciferases is inhibited in the presence of detergent in a concentration dependent manner at detergent concentrations of less than 1 % are particularly surprising in light of the previous dogma in the art that Gaussia and Metridia luciferase activity is sodium dependent and/or sensitive to cation concentration and is not inhibited by non-ionic detergents up to or above 2%,
  • the bioluminescent signal generated by the luciferase can be prolonged during a "glow" phase that begins some minutes following addition of the luciferase substrate.
  • the bioluminescent signal can be prolonged during a phase that begins between about 3 minutes and about 15 minutes after addition of the substrate.
  • the bioluminescent signal generated by the luciferase may be such that starting from 10 minutes after initiation of the luciferase-catalysed reaction, the bioluminescent signal decays with a half-life of more than 20 minutes or more than 30 minutes.
  • the luciferase substrate may be a constituent of the reagent composition or may be independent. Where the luciferase substrate is not a constituent of the reagent composition, the substrate may be added to the sample containing luciferase either before, after or at the same time as addition of the reagent composition.
  • luciferase-based reporter assay systems employ two buffers, a lysis buffer and an assay buffer (collectively referred to herein as "assay reagents").
  • the lysis buffer typically comprises components for lysing the cells containing the luciferase to be assayed while the assay buffer typically contains, inter alia, the substrate and any required cofactors for the luciferase reaction.
  • Reagent compositions of the present invention are typically in the form of cell lysis buffers.
  • lysis buffers in accordance with the present invention effectively provide shorter lysis times than is possible with currently available buffers.
  • the present inventors suggest that these buffers provide a suitable environment for promoting or facilitating the folding or conversion of the luciferase to be assayed following cell lysis into an active state or conformation from an inactive state or conformation, or into a more active state or conformation from a less active state or conformation.
  • reagent compositions of the invention may be in the form of a combined lysis and assay buffer such that only a single buffer composition is required to lyse cells, potentially directly within the medium in which the cells are cultured, and initiate the luciferase-catalysed reaction.
  • Reagent compositions in accordance with the present invention may typically be aqueous solutions, or alternatively may be in solid or dry form such as lyophilised. Whether aqueous or lyophilised, reagent compositions of the invention may be provided either comprising all constituents pre-mixed or as a combination of constituents to be mixed prior to use. The reagent composition may be used directly in an assay system for the determination of luciferase amount and/or activity, or may be reconstituted, dissolved, diluted or otherwise treated either chemically or physically such that the composition is capable of performing the desired function. Reagent compositions of the present invention are applicable to determining the amount and/or activity of any luciferase in a sample.
  • the luciferase may be a naturally occurring enzyme or modified enzyme.
  • a naturally occurring luciferase may be derived from any one of a number of bioluminescent organisms, typically from the light organ thereof. Such organisms include but are not limited to bioluminescent bacteria, protozoa, coelenterates, molluscs, fish, flies, crustaceans and beetles.
  • luciferases may be categorised according to the substrate utilised by the enzyme.
  • One group of luciferases such as those of fireflies and click beetles utilise luciferin (D-luciferin).
  • a second group of luciferases such as those of the marine organisms Renilla, Gaussia, Pleuromamma, Metridia and Cypridina utilise coelenterazine.
  • Other luciferases such as Vargula luciferase, use a different substrate.
  • the reagent compositions of the present invention are applicable to use with luciferases using luciferin or coelenterazine or any other substrates.
  • the reagent compositions of the invention are similarly applicable to use with either intracellular or secreted iuciferases.
  • Firefly and Renilla luciferases are intracellular in their natural state, whereas Gaussia and Metridia luciferases are secreted in their wild-type state.
  • Gaussia luciferase is of particular interest as it has been shown to yield a bioluminescent signal strength higher than that achievable with Renilla luciferase and is the smallest known luciferase.
  • Other secreted luciferases have also been shown to yield strong signal strength, for example Meritidia longa luciferase.
  • the luciferase may be a variant or derivative of a naturally occurring luciferase.
  • the present invention finds particular application in reactions and assays involving modified, non-secreted forms of luciferases that are secreted in their native form.
  • a naturally secreted luciferase may be modified using standard molecular biological techniques by removal of signal sequences and/or fusion to an intracellular polypeptide such that the enzyme is no longer secreted but remains intracellular.
  • luciferase polypeptide sequence may be altered, for example, to alter one or more amino acids in the luciferase polypeptide sequence to modulate expression and/or solubility of the enzyme in a cell culture system of choice.
  • modulation may be an increase or decrease in expression and/or solubility, depending on the requirements of the particular application in which the luciferase, and the reagent compositions of the invention are to be employed.
  • Luciferases containing destabilising elements have shortened half-lives and are expressed at lower steady-state levels than luciferases which do not contain such elements.
  • Suitable protein destabilising elements include PEST sequences (amino acid sequences enriched with the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T)), a sequence encoding an intracellular protein degradation signal or degron, and ubiquitin.
  • P proline
  • E glutamic acid
  • S serine
  • T threonine
  • the enhanced sensitivity attained with reagent compositions of the present invention are particularly advantageous for use with destabilized luciferases given the lower steady state expression levels of such luciferases. Any suitable method for destabilising a protein is contemplated herein.
  • luciferase may also be modified by, for example, the addition of sequences such as poly(A) tails, transcriptional or translational enhancers, and/or adapting codon usage in the encoding polynucleotide sequence for a particular expression system.
  • sequences such as poly(A) tails, transcriptional or translational enhancers, and/or adapting codon usage in the encoding polynucleotide sequence for a particular expression system.
  • codon usage in the luciferase polynucleotide may be optimised for insect or human cells respectively. Approaches for codon usage adaptation and optimisation for different species are well known to those skilled in the art.
  • luciferase polypeptide or polynucleotide sequences are also well known to those skilled in the art.
  • restriction enzyme cleavage sites may be introduced into the polynucleotide, or the luciferase polypeptide may be fused or conjugated with a second polypeptide of different function such as a selectable marker (e.g. antibiotic resistance).
  • a selectable marker e.g. antibiotic resistance
  • luciferases that are particularly amenable to use in accordance with the invention, as well as predict the modifications that may be made to a secreted luciferase the render the luciferase non-secreted.
  • luciferases that are secreted in their native form typically comprise cysteine residues that form disulphide bridges in the mature active conformation of the protein.
  • the cysteine residues may be arranged in spacing patterns that are repeated within the amino acid sequence such that they can be predicted to form intramolecular disulphide bonds.
  • Such luciferases typically show reduced activity when expressed intracellularly.
  • homologous luciferases sharing one or more of these characteristics of naturally secreted luciferases are particularly suitable for use in accordance with the present invention.
  • Suitable luciferases are readily obtainable by those skilled in the art using known techniques.
  • the luciferase may be directly obtained from the light organ(s) of the bioluminescent organism.
  • the luciferase may be obtained from cultured cells, for example bacteria, yeast, insect cells or mammalian cells which have been transformed with nucleic acids encoding the luciferase.
  • luciferases When using secreted luciferases in in vitro reporter assays, a sample of the cell culture medium, rather than a cell lysate, is typically taken for measurement of luciferase activity. Whilst advantageous in some applications (e.g. repeated measurements from the same cells), secreted luciferases are not appropriate for other applications. In particular, the secreted luciferase accumulates in the cell culture medium such that rapid changes in gene expression can not be monitored accurately. Mutant, non-secreted forms of these luciferases (preferably containing destabilizing elements) overcome this limitation.
  • One particular modified luciferase described herein is a modified Gaussia luciferase in which the 14 amino acid N-terminal signal peptide is deleted thereby generating a non-secreted luciferase.
  • a second modified luciferase described herein is a modified Metridia luciferase in which the 17 amino acid N-terminal signal peptide is deleted thereby generating a non-secreted luciferase.
  • Other secreted luciferases can be similarly modified for intracellular expression, particularly in eukaryotes, using a variety of methods well known to those skilled in the art.
  • reagent compositions of the present invention find application in reporter assay systems based on a luciferase, either intracellular or secreted, which utilises coelenterazine as a substrate or in dual luciferase reporter assays in which at least one of the luciferases utilises coelenterazine as a substrate and/or is secreted in its native form.
  • luciferase activity can be detected and measured by any one of a number of methods well known to those skilled in the art, including but not limited to using a luminometer, a scintillation counter, a photometer such as a photomultiplier photometer or photoemulsion film, or a charge-coupled device (CCD).
  • Reagent compositions of the invention provide improved kinetics of bioluminescence in luciferase reactions and offer advantages over the prior art. As such these compositions may be used in luciferase assays according to the invention, in preparing assay reagents and test kits according to the invention, and in standards and controls for assays and kits according to the invention.
  • kits for carrying out assays of luciferase activity such kits containing reagent compositions in accordance with the invention as described herein.
  • Kits of the invention comprise, in one or more physical containers and typically packaged in a convenient form to facilitate use in luciferase assays, suitable quantities of reagent compositions or constituents thereof for carrying out luciferase assays. Multiple reagent compositions, or various constituents of reagent compositions may be combined, for example in aqueous solution or lyophilised, in a single container or in multiple containers.
  • Kits of the invention typically also comprise controls and standards to ensure the reliability and accuracy of assays carried out in accordance with the invention. Suitable controls and standards will be known to those skilled in the art..
  • Firefly luciferase and Renilla luciferase are the most commonly used luciferases in commercial luciferase-based reported systems. Although the secreted Gaussia luciferase and secreted Metridia luciferase provide higher sensitivity than firefly or Renilla luciferase, secreted luciferases accumulate in the culture medium and therefore are not ideal for reporter studies aimed at measuring temporal changes in gene expression.
  • the inventors cloned the Gaussia luciferase coding sequence into various RapidReporter plasmids (GeneStream) using a PCR strategy that deleted the 15 amino acid N-terminal signal peptide and fused the remaining Gaussia luciferase cDNA to N-terminal and C- terminal coding sequences that included sequences encoding protein destabilizing elements.
  • the modified Gaussia luciferase is not secreted when expressed in mammalian cells.
  • modified luciferase enzyme The precise intracellular location of the modified luciferase enzyme is assumed to be cytoplasmic.
  • mammalian cells transfected with plasmids expressing either the native Gaussia luciferase (hereafter referred to as "secreted Gaussia luciferase") or the modified Gaussia luciferase (hereafter referred to as "non-secreted Gaussia luciferase”) were lysed in the presence of a variety of lysis buffer compositions and bioluminescence measured.
  • the inventors cloned the Metridia luciferase coding sequence into various RapidReporter plasmids (GeneStream) using a PCR strategy that deleted the 17 amino acid N-terminal signal peptide and fused the remaining Metridia luciferase cDNA to N-terminal and C- terminal coding sequences that included sequences encoding protein destabilizing elements.
  • the modified Metridia luciferase is not secreted when expressed in mammalian cells.
  • modified luciferase enzyme The precise intracellular location of the modified luciferase enzyme is assumed to be cytoplasmic.
  • mammalian cells transfected with plasmids expressing either the native Metridia iuciferase (hereafter referred to as "secreted Metridia luciferase") or the modified Metridia luciferase (hereafter referred to as “non-secreted Metridia luciferase”) were lysed in the presence of a variety of lysis buffer compositions and bioluminescence measured.
  • the bioluminescent signal strength was very low, particularly in samples in which luciferase activity was measured shortly after cell lysis. Only after some hours of incubation in lysis buffer did the modified luciferase gain high activity.
  • the non-secreted Gaussia and Metridia luciferases adopt a less active conformation intracellular ⁇ but can adopt an active conformation following cell lysis, albeit slowly.
  • the inventors then tested a variety of modifications to the lysis buffer components in an attempt to develop a formulation enabling shorter incubation periods and in which the luminescent signal comprises a stronger flash phase (higher sensitivity) and a more stable glow phase (rate of decay of the signal is reduced); in particular providing an environment promoting the conversion of the luciferase from an inactive conformation to an active conformation following cell lysis.
  • HeLa cells were transiently transfected with a plasmid expressing native, secreted Gaussia luciferase and 24 hrs later the conditioned medium was collected.
  • Wells of a 96-well plate were loaded with 20ul of conditioned medium and assayed for luciferase activity by injecting 60uI of an assay buffer comprising 26uM Cz; pH 8.1 plus the salt as indicated in Fig. 1 . Results were expressed as a % of the light units obtained in the absence of salt ( Fig. 1 ).
  • HeLa cells stably expressing non-secreted and destabilized Gaussia luciferase were plated onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 30 ⁇ l of lysis buffer per well.
  • the lysis buffer (LB) comprised; 25 mM Tris pH 7.8; 0.1% NP40; 1 mM EDTA plus NaBr at concentrations of (from left to right) 0mM, 150mM, 0mM and 75mM.
  • Luciferase activity was assayed by injecting 30uI of an assay buffer (AB) comprising 25 mM Tris pH 7.8; 40 uM Cz plus NaBr at concentrations of (from left to right) 0mM, 0mM, 150mM and 75mM. Thus, the final concentration of NaBr was 75mM in all samples, except the no salt controls. Results were expressed as relative light units (RLU) (See Fig. 2 ).
  • RLU relative light units
  • Fig. 2 show that the beneficial effect of NaBr with secreted Gaussia luciferase ( Fig. 1 ) also applies to the non-secreted and destabilized Gaussia. Moreover, the data show that it is advantageous to include NaBr in the lysis buffer (LB) as opposed to the assay buffer (AB). It can be seen that NaBr enhances activity of non-secreted Gaussia luciferase, especially when included in lysis buffer.
  • HeLa cells stably expressing non-secreted and destabilized Gaussia luciferase were plated onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ⁇ l of lysis buffer per well.
  • the lysis buffer comprised; 25 mM Tris pH 8.1, 1 mM EDTA; 0.1% NP40; 63.4 uM Na-Oxalate; 5% Glycerol plus the concentration and type of salt indicated in Fig. 3 .
  • Luciferase activity was assayed at 40 min after addition of the lysis buffer(by injecting 60 ⁇ l of an assay buffer comprising 25 mM Tris pH 8.1; 1 mM EDTA, 2 mM Ascorbate; 26 uM Cz. Results were expressed as a % of the light units obtained in the absence of salt (see Fig. 3 ). Both salts increased light output compared to no salt. However NaBr provided a superior enhancement to NaCI and enhancement was found to have some concentration dependency.
  • HeLa ceils stably expressing either non-secreted and destabilized Gaussia or non- secreted and destabilized Metridia luciferase were plated onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of lysis buffer per well.
  • the lysis buffer comprised; 25 mM Tris pH 8.1; 0.1% NP40; 63.4 ⁇ M Na-Oxalate; 5% Glycerol plus the concentration and type of salt indicated in Fig. 4 .
  • Replicate samples were assayed for luciferase activity after 30 minutes following addition of the lysis buffer by injecting 60 ⁇ l of assay buffer.
  • the assay buffer comprised 25 mM Tris pH 7.75; 2 mM Ascorbate; 24 uM Cz. Results were expressed as a % of the light units obtained in the absence of salt (see Fig. 4 ).
  • NaBr provided a superior enhancement to NaCI with non-secreted and destabilized Metridia luciferase.
  • the enhancement was found to have some concentration dependency.
  • Luciferase assays were performed as described in Example 5 with either non-secreted and destabilized Gaussia luciferase or non-secreted and destabilized Metridia luciferase. Two experiments are shown as separate graphs, with results shown as relative luminescence in counts per second (CPS) (see Figs. 5A and 5B ). Within each experiment, the lysis buffer and assay buffer differed only in the amount and type of salt added as indicated. Replicate samples were analysed at 120 min after addition of the lysis buffer (LB) by injecting 60 ⁇ l of assay buffer (AB).
  • the signal strength is then lowest in the absence of salt. A higher signal strength was noted by including salt in either the lysis buffer or assay buffer. Furthermore, the bromide salt was superior to the chloride salt for both luciferases. Of the four treatment groups, the highest signal strength was seen when the bromide was included in the lysis buffer suggesting that in some circumstances it may be preferable to include the bromide in the lysis buffer rather than the assay buffer.
  • Luciferase assays were performed as described in Example 4. All salts were present at 150 mM in lysis buffer,
  • Luciferase assays were performed as described in Example 7, except using the indicated concentrations of salt and EDTA in the lysis buffer. Replicate samples were assayed for luciferase activity at 20, 30, 40 and 90 min after addition of lysis buffer. Results were expressed as a % of the light units obtained after 20 min lysis in the same lysis buffer (see Fig. 7 ). In the absence of salt and EDTA1 a dramatic increase in luciferase activity was evident between 20 and 90 min after the onset of cell lysis. A far more stable (and therefore more desirable) level of luciferase activity was achieved by addition of either EDTA or NaBr alone.
  • Luciferase assays were performed as described in Example 8, except using lysis buffer containing 150 mM NaBr and the indicated concentration of EDTA. Two experiments are shown as separate graphs, with results shown as relative light units (RLU) (see Figs. 8A and 8B ). Within each experiment, the same assay buffer was used for all samples and the lysis buffer differed only in the amount of EDTA as indicated. Replicate samples were assayed for activity of non-secreted Gaussia luciferase at 30 or 120 min after addition of lysis buffer. The benefit of 1 mM EDTA vs. no EDTA is shown in the previous graph. This graph demonstrates the benefit of higher amounts of EDTA in terms of both signal intensity and the stability of the luciferase signal over time in lysis buffer.
  • RLU relative light units
  • Luciferase assays were performed as described in Example 9, except using the indicated type and concentration of chelator and a 30 min lysis period. The results show that both EDTA and EGTA provide a concentration-dependent beneficial effect on signal intensity (see Fig. 9 ).
  • Luciferase assays were performed with non-secreted and destabilized Metridia luciferase as described in Example 5, except the lysis buffer contained no salt and but contained the indicated type and concentration of chelators. Replicate luciferase assay were carried out after a 30 or 120 min lysis period. Results from the 30 min time point were expressed as a % of the light units obtained in the absence of chelator (see Fig. 10A ) and results from the 120 min time point were expressed as % of the light units obtained with the same lysis buffer at 30 min (see Fig 10B ). The results show that EDTA, CDTA and EGTA provide a concentration-dependent beneficial effect on signal intensity and additionally reduce the effect if lysis time.
  • Flasks of HeLa cells transiently expressing secreted Gaussia or secreted Metridia luciferases were incubated overnight. Aliquots of conditioned media were removed and diluted 1:10 into fresh medium (RPMI + 10% fetal calf serum) with or without 5 mM EDTA. After 30 min, 80 ul aliquots were assayed for luciferase activity by injecting 20 ⁇ l medium containing 48 ⁇ M Cz. Results were expressed as the % of the light units obtained in the absence of EDTA (see Fig. 11A ). A second experiment was performed in the same manner except that the incubation time was increased to 90 min and some samples received assay buffer that additionally contained 5 mM EDTA (see Fig. 11B ).
  • HeLa cells expressing Firefly or Renilla luciferases were plated into 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ⁇ l of lysis buffer containing 25 mM Tris pH 8.1; 0.1% NP40; 63.4 ⁇ M Na-Oxalate; 5% Glycerol, 150 mM NaCl plus the concentration of EDTA in Fig. 12 . Replicate samples were assayed for luciferase activity at 30 min by injecting 30 ⁇ l of Firefly assay buffer Il containing Firefly substrate followed by 30 ul of Promega's 'Stop and Glo" buffer containing Renilla substrate.
  • Results are expressed as a % of the light units obtained in the absence of EDTA (see Fig. 12 ).
  • the results indicate that the addition of EDTA to Renilla and firefly luciferases does not provide the same beneficial effect on signal intensity as is observed with non-secreted Gaussia or Metridia luciferases. In fact, the addition of EDTA clearly decreases the activity of Firefly luciferase.
  • HeLa cells expressing destabilised, non-secreted Gaussia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. Three experiments are shown as separate graphs ( Figs. 13A, 13B and 13C ). Within each experiment, the same assay buffer was used for all samples and the lysis buffer differed only in the type of detergent used (0.1 % in lysis buffer unless otherwise indicated). The anionic detergents, SDS and DOC do not perform well with Gaussia luciferase, presumably because they inhibit luciferase activity.
  • HeLa cells expressing destabilised, non-secreted Gaussia or non-secreted Metridia luciferases were plated in equal aliquots onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ⁇ l of lysis buffer. The detergent type and concentration was varied as indicated.
  • Luciferase assays were carried out by injecting 60 ⁇ l of assay buffer comprising 25 mM Tris pH 8.1; 1 mM EDTA, 2 mM Ascorbate; 24 ⁇ M Cz Two experiments are shown, in the first the lysis buffer also contained 5% glycerol, 64uM sodium oxalate, 150mM NaBr, 25mM Tris pH 8.5, 5mM EDTA, 0.6 mM reduced glutathione, 0.4mM oxidised glutathione, 75mM urea (v6) and luciferase activity was assayed at 40 min.
  • assay buffer comprising 25 mM Tris pH 8.1; 1 mM EDTA, 2 mM Ascorbate; 24 ⁇ M Cz Two experiments are shown, in the first the lysis buffer also contained 5% glycerol, 64uM sodium oxalate, 150mM NaBr, 25mM Tris pH 8.5, 5mM EDTA
  • the lysis buffer also contained 2 5mM Tris pH 8.1, 63.4 uM Na-oxalate, 150mM NaBr, 5% Glycerol and the luciferase activity was assayed at 120 min. Results were expressed as the % of the light units obtained using 0.1% NP40 (see Figs. 14A and 14B ).
  • the results from the first experiment indicated that 0.1% SDS blocks enzymatic activity and that 0.1% CHAPS is insufficient to lyse the cells, therefore different concentration ranges of these detergents were used in the second experiment (see Fig 14B ),
  • HeLa cells expressing secreted Gaussia or secreted Metridia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. Medium was removed and diluted 1:10 into fresh medium containing the type and concentration of detergent indicated in Figs. 15A and 15B . After 30 min, 80 ⁇ l were removed, 20 ⁇ l of medium containing 48uM Cz added and the samples assayed for luciferase (see Figs 15A and 15B ) Results were expressed as the % of the light units obtained in the absence of detergent.
  • HeLa cells expressing destabilised, non-secreted Gaussia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. Two experiments are shown as separate graphs (see Figs. 16A and 16B ). Within each experiment, the same assay buffer was used for all samples and the lysis buffer differed only in the % NP40. The highest signal was seen with 0.1 % and signal strength declined with increasing amounts of detergent. It can be seen that luciferase activity is inhibited by detergent in a concentration-dependent manner.
  • Luciferase assays were performed as described in Example 9, except using lysis buffer with the indicated pH. EDTA was present at 1mM except where indicated. Four experiments are shown as separate graphs (see Figs. 18A, 18B, 18C and 18D ). Lysis times were 40 minutes for A and B, 30 minutes for C; and in D, and replicate samples were assayed for Gaussia luciferase activity at 30 or 120 minutes after addition of lysis buffer. These data demonstrate the benefit of higher pH in terms of both signal intensity and the stability of the luciferase signal over time in lysis buffer. The combination of high pH and high EDTA was particularly effective.
  • HeLa cells expressing destabilised, non-secreted Gaussia or non-secreted Metridia luciferases were plated in equal aiiquots onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ⁇ l of lysis buffer.
  • the lysis buffer comprised 63.4uM Na-oxalate, 5% Glycerol, 150mM NaBr and 25mM Tris at the indicated pH.
  • Luciferase assays were carried out at 30 min by injecting 60 ⁇ l of assay buffer comprising 25 mM Tris pH 7.75; 2mM Ascorbate; 24 uM Cz. Results were expressed as the % of the light units obtained at pH 8.5 (see Fig. 19 ).
  • Luciferase assays were performed as described in Example 18, with EDTA at 5 mM and pH 8.5.
  • the lysis buffer also contained the indicated concentrations of reduced (red) and oxidised (ox) glutathione.
  • Replicate samples were assayed for Gaussia luciferase activity at 20 or 135 minutes after addition of lysis buffer.
  • Luciferase assays were performed as described in Example 21, using the indicated total amounts of glutathione and ratio of reduced:oxidized. Replicate samples were assayed for Gaussia activity at 20 or 60 minutes after addition of lysis buffer. These data demonstrate that at all concentrations and ratios tested, the presence of glutathione in lysis buffer increases the rate at which the luciferase acquires its maximum activity during the cell lysis step. At the higher concentrations of reduced glutathione, a somewhat reduced signal was seen at 60 minutes (see Fig. 21 ).
  • HeLa cells transiently expressing either non-secreted Gaussia luciferase or non-secreted Metridia iuciferase were plated onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ⁇ l of lysis buffer per well, comprising 25 mM Tris pH 8.1, 63.4 uM Na- oxalate, 0.1% NP40, 5% Glycerol and either no glutathione or a combination of 0.6 mM reduced glutathione and 0.4mM oxidised glutathione.
  • Replicate samples were assayed for luciferase activity at 30 min after addition of lysis buffer by injecting 60 ul of an assay buffer comprising 25 mM Tris pH 7.75, 0.6 mM reduced glutathione, 0.4mM oxidised glutathione, 1 mM EDTA, 2 mM Ascorbate, 24 uM Cz Results were expressed as the % of the light units obtained in the absence of glutathione for the same luciferase (see Fig. 22 ).
  • Luciferase assays were performed as described in Example 22 except using a single concentration and ratio of additive (glutathione; 1.2 mM reduced, 0.8 mM oxidised) in either the lysis buffer and/or assay buffer or neither. Two experiments are shown as separate graphs (see Figs. 23A and 23B ). Replicate samples were assayed for Gaussia luciferase activity at 15 or 60 minutes after addition of lysis buffer. These data demonstrate that the presence of glutathione in lysis buffer but not assay buffer increases the signal attained after a short (15 min) lysis period.
  • Luciferase assays were performed as described in Example 23 with the glutathione in both lysis buffer and assay buffer. Additionally, the lysis buffer contained the indicated concentrations of urea. Replicate samples were assayed for Gaussia luciferase activity at 15 or 60 minutes after addition of lysis buffer and the 60 min data were expressed as a % of the signal at 15 mins. All lysis buffers showed an increase in signal during this time. However, the increase was less pronounced with 50-100 mM urea suggesting that maximum activity can be reached sooner under these conditions (see Fig. 24 ).
  • HeLa cells expressing destabilised, non-secreted Gaussia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. The same assay buffer was used for all samples. Replicate samples were assayed for luciferase activity at 20 or 120 minutes after addition of lysis buffer containing 5% glycerol, 64 ⁇ M sodium oxalate, 0.1% NP40, 150mM NaBr plus either 25mM Tris pH 8.1, 1mM EDTA (v3) or 25mM Tris pH 8.5, 5mM EDTA, 0.6mM reduced glutathione, 0.4mM oxidised glutathione, 75mM urea (v6). Whereas v3 lysis buffer achieved only -50% maximal activity after 20 mins lysis, the v6 lysis buffer achieved 100% activity within 20 mins (see Fig.25 ).
  • HeLa cells expressing destabilised, non-secreted Metridia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of lysis buffer containing 5% glycerol, 63.4 ⁇ M sodium oxalate, 0.1% NP40, plus 25mM Tris at the indicated pH, the indicated salt at 150 mM and, where indicated, 1 mM EDTA or 0.6 mM reduced glutathione and 0.4mM oxidised glutathione (GSH/GSSG)
  • Replicate samples were assayed for luciferse activity at 30 or 120 min after addition of lysis buffer by injecting 60uI of assay buffer containing 25mM Tris pH 7.75, 2 mM ascorbate, 24 uM Cz. Results were expressed as the % of the light units obtained at 30 min lysis (see Fig. 26 ).
  • HeLa cell expressing non-secreted Gaussia or Renilla luciferases were plated into 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ⁇ l of GeneStream's v6 (as Example 26) lysis buffer or Promega's passive lysis buffer (PLB) per well. Luciferase activity was assayed by injecting 60 I of assay buffer comprising 25 mM Tris pH 7.75; 0.6 mM reduced glutathione, 0.4 mM oxidised glutathione, 1 mM EDTA, 2 mM Ascorbate, 24 uM Cz (see Fig. 27A ).
  • Fig. 27A The results in Fig. 27A indicate that GeneStream's v6 (GSv6) buffer is at least as effect as Promega's passive lysis buffer (PLB) for use with Renilla luciferase but is superior (10-fold higher signal) to PLB for use with non-secreted Gaussia luciferase.
  • PLB Promega's passive lysis buffer
  • the data shown in Fig. 27B shows that Renilla luciferase gives the strongest signal with live cells.
  • the data combined indicates that Gaussia luciferase adopts a less active form at when expressed intracellularly.
  • HeLa cells expressing destabilised, non-secreted Gaussia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight (see Fig. 28 ).
  • PA 1 luciferase assay performed with Promega's dual luciferase assay kit according to the manufacturer's instructions (i.e.
  • Example 29 The method of Example 29 was followed except that the kinetics of light emission were measured.
  • the data show that compared to a prior art "glow" buffer, the compositions that are the subject of the present invention provide a more stable glow signal and higher sensitivity (see Figs. 29A and 29B ).
  • HeLa cells expressing destabilised, non-secreted Gaussia and non-secreted Metridia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ⁇ l of lysis buffer. Replicate samples were assayed for luciferase activity at 20, 30, 40, 60, 90 or 120 min after addition of lysis buffer.
  • the lysis buffers used were GeneStream's v3 and v6 (as Example 26), Promega's passive lysis buffer (PLB) and a generic standard lysis buffer (STD-LB) containing 25 mM Tis pH 7.7; 1% Triton X100; 10% glycerol.
  • compositions that are the subject of the present invention provide a higher and more stable signal intensity for both non-secreted Gaussia luciferase and non-secreted Metridia luciferase.
  • data in Fig 30B demonstrates that reduced lysis time can be employed when using the composition of the present invention compared to prior art compositions.
  • Luciferase-based gene reporter assay kits according to the invention are provided in 2- parts designed for 20 ⁇ l lysis buffer and 60 ⁇ l assay buffer per well of a 96-well plate.
  • HeLa cells expressing a modified non-secreted Gaussia luciferase are cultured in 96-well plates, lysed with lysis buffer (20 ⁇ I) after removing any medium and are assayed for luciferase activity using a Wallac Victor 3 luminometer (Perkin Elmer) in flash reactions following injection of assay buffer (60 ⁇ I).

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